Infections with human herpesviruses are endemic in the population with some herpesviruses causing severe disease, especially in immune impaired individuals and newborns. The increased immunocompromised population has created an unmet medical need for antivirals against herpesviruses. The goal of the proposed project is to gain a better understanding of DNA cleavage and packaging in herpes simplex virus (HSV) through the analysis of two viral proteins, UL25 and UL28, that are required for this process. The specific hypothesis behind the proposed research is that UL25 and UL28 are required for packaging DNA into HSV capsids at separate stages in the cleavage packaging reaction. The UL28 protein is required for cleavage of concatemeric DNA into unit length molecules while UL25 serves an essential function in the production of DNA containing capsids. Although, the focus will be on UL25 and UL28 the goal is to characterize the cleavage/packaging process using genetic and biochemical approaches to examine how mutations in the UL25 and UL28 genes affect the interaction of these proteins with: (i) DNA, (ii) capsid proteins, (iii) cleavage/packaging proteins and (iv)host cell proteins. Specific goals of the project are to: 1. Determine the capsid location, DNA binding specificity and protein-protein interactions of UL25. 2. Identify functional domains of UL28 that are important for capsid incorporation, binding viral DNA packaging sites and for interaction with terminase (UL15) and portal protein (UL6) by characterizing a series of HSV-1 UL28 mutants that we have isolated; and map the second site mutation for a revertent of one of the lethal UL28 linker- insertion mutants since marker rescue and DNA sequencing have demonstrated that the mutation does not map to the UL28 gene. 3. Examine the interaction of UL25 and UL28 with viral and cell proteins using proteomic and biochemical approaches in order to identify essential protein-protein interactions and protein complexes that are involved in the cleavage/packaging reaction. These studies aim to elucidate the mechanism underlying HSV DNA cleavage and packaging and may suggest novel targets for the development of antivirals.